To purify mouse SGK1 protein, Flag-SGK1 was transfected into HEK293T cells for 48h, cells were then lysed using RIPA buffer, and lysate was incubated with M2-Flag magnetic beads from SIGMA following its IP protocol. 3XFlag peptide was used to elute Flag-SGK1 from the beads. To purify AMPKα1 protein, pcDNA3-AMPKα1 with His tag was transfected into HEK293T cells for 48h and purified using the Pierce HisPur Ni-NTA Purification Kit (Thermo Scientific, Prod# 88227). SGK1(0.5 μg) and AMPK α1 (1 μg) or AMPKα2β1γ1 (1 μg) complex were incubated in a 25ul reaction containing 5 μL 5x kinase buffer, 1 mM DTT, and 500 μM ATP for 15 min.
Pierce hispur ni nta purification kit
Manufactured by Thermo Fisher Scientific
The Pierce HisPur Ni-NTA Purification Kit is a product designed for the purification of histidine-tagged (His-tagged) recombinant proteins. It contains pre-packed Ni-NTA resin columns and all the necessary buffers for efficient protein purification.
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Lab products found in correlation
2 protocols using pierce hispur ni nta purification kit
1
Purification and Kinase Assay of SGK1 and AMPK
2
Purification and Kinase Assay of SGK1 and AMPK
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To purify mouse SGK1 protein, Flag-SGK1 was transfected into HEK293T cells for 48h, cells were then lysed using RIPA buffer, and lysate was incubated with M2-Flag magnetic beads from SIGMA following its IP protocol. 3XFlag peptide was used to elute Flag-SGK1 from the beads. To purify AMPKα1 protein, pcDNA3-AMPKα1 with His tag was transfected into HEK293T cells for 48h and purified using the Pierce HisPur Ni-NTA Purification Kit (Thermo Scientific, Prod# 88227). SGK1(0.5 μg) and AMPK α1 (1 μg) or AMPKα2β1γ1 (1 μg) complex were incubated in a 25ul reaction containing 5 μL 5x kinase buffer, 1 mM DTT, and 500 μM ATP for 15 min.
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